Purpose :
The choice of plasma or serum samples for the quantification of Vascular Endothelial Growth Factor (VEGF-A) in patients with retinal neovascularization remains controversial. Blood drawing procedures activate coagulation thus leading to varying concentrations of VEGF in serum compared to plasma or even among different anticoagulant molecules. Additionally, because of the fragile situation in pre-term babies and the small volume of blood that can be taken, little information is available about VEGF-A levels in blood samples of term and pre-term infants. In this study, the VEGF-A Status in blood samples, namely serum, plasma and other blood compartments, of healthy adults as well as term and pre-term infants was assessed.

Methods :
Venous blood samples of healthy adults (n=10) and cord blood samples of term (n=13) as well as preterm (n=26) infants were collected. Serum, serum from recalcified citrate blood (rS), Citrate plasma (CB_P), Citrate-Theophylline-Adenine-Dipyridamol (CTAD) plasma (CTAD_P) and platelets suspensions were obtained. The VEGF-A and Platelet Factor 4 (PF-4) concentration was measured by Enzyme-linked Immunosorbent Assay (DuoSet ELISA, Fa. R&D Systems) or AlphaLISA immunoassay (Fa. Perkin Elmer). Additionally, citrate plasma and rS samples of 7 infants with ROP were also analysed, two of them before and after treatment with intravitreal bevacizumab (IVB).

Results :
VEGF-A levels in CB_P samples of healthy adults and term and pre-term infants were between below the detectable level and 220 pg/ml for both ELISA and AlphaLISA assays. PF-4 concentrations in the same samples were between 0,15 – 4,2 µg/ml. No significant differences between the concentrations of VEGF-A in CB_P and CTAD_P were found. VEGF-A as well as PF4 levels in serum and recalcified serum from citrate blood (rS) samples correlated significantly. In CB_P samples of infants with ROP VEGF-A values ranged from 6 to 200 pg/ml. Concentrations of PF-4 lay between 0,1 and 3,3 µg/ml. VEGF-A concentrations decreased following treatment.

Conclusions :
The influence of retinal neovascularization on the peripheral blood concentrations of VEGF-A can be best addressed by measuring free circulating VEGF-A in plasma samples. Any kind of platelet activation during sampling or sample processing needs to be avoid, which can be monitored by PF4 measurements.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.